Cholera toxin subunit B (CTxB) binds to molecules presented on the surface of host intestinal epithelial cells. After the initial binding event, the holotoxin invades the cells, hijacking the cellular machinery to cause the profuse diarrhea that characterizes cholera infection. A complete understanding of the mechanistic details of cholera toxicity is necessary to facilitate the development of novel strategies to combat cholera outbreaks. While the ganglioside GM1a is widely described as the single receptor recognized by CTxB, recent data from our lab and others implies the existence of additional cholera toxin binding partners, which may be glycoproteins. The broad goals of this proposal are to identify novel CTxB binding partners and discover whether they function in cholera intoxication. The goal of Aim 1 is to identify cell surface molecules in T84 cells that are recognized by CTxB, making use of a novel photocrosslinking technique and modern mass spectrometry methods, along with traditional affinity purification approaches. Additional CTxB binding partners could play an auxiliary role, assisting in formation of the canonical CTxB-GM1a complex, or they might function as alternative receptors, enabling cholera toxin to bind cells in the absence of GM1a. The goal of Aim 2 is to distinguish between these possibilities, by using shRNAs to transiently knockdown expression of these alternative binding partners and glycosylation inhibitors to interfere with their post- translational modification. The results of these experiments will allow us to determine if the novel receptor or receptors are required for CTxB binding to T84 cells. While alternative binding partners might facilitate CTxB binding, a more critical question is whether they function in internalization of the toxin. The results of the immunofluorescence microscopy experiments and chloride ion secretion assays, proposed in Aim 3, will reveal whether previously unidentified CtxB receptors facilitate the process of cholera intoxication. This project challenges the existing paradigm that GM1a is both necessary and sufficient for CTxB binding and internalization. Along with the impact on our understanding of cholera disease progression, the results of the proposed experiments will be important for understanding and distinguishing among different mechanisms of endocytosis. Furthermore, because CTxB is commonly used to identify and visualize lipid rafts, a complete knowledge of CTxB binding partners will be vital to defining the composition and characteristics of membrane microdomains. PUBLIC HEALTH RELEVANCE: The bacteria Vibrio cholerae produce a toxin that binds to and invades human intestinal cells, causing the profuse diarrhea that characterizes cholera infection. The cholera toxin has long been believed to interact with a molecule known as GM1a, but our data suggest that the toxin may also interact with an additional molecule or molecules. We propose to identify the additional molecules with which cholera toxin interacts and to determine whether these interactions are critical to disease progression.